14-3-3 Binding molecules as sensitizers for anticancer therapies

ABSTRACT

Methods are provided for enhancing the death of a neaplastic cell comprising the administration of a therapeutically effective concentration of a 14-3-3 antagonist and at least one antineoplastic therapeutic agent. The methods of the invention find use in improving the clinical outcome of a mammal having a neoplastic disorder and comprises administration to a mammal in need thereof at least one antineoplastic therapeutic agent in combination with a 14-3-3 antagonist. Further provided are pharmaceutical compositions having a therapeutically effective amount of a 14-3-3 antagonist and an antineoplastic therapeutic agent. Also provided are methods for identifying agents that selectively inhibit an interaction between a 14-3-3 polypeptide and a 14-3-3 ligand.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This research was funded in part by the National Institute ofHealth, contract number GM53165.

FIELD OF THE INVENTION

[0002] The present invention is directed to a method for enhancing thedeath of a neoplastic cell comprising the administration of atherapeutically effective concentration of a 14-3-3 antagonist and anantineoplastic therapeutic agent. The invention is further directed tomethods of identifying 14-3-3 antagonists useful in the treatment ofneoplastic disorders.

BACKGROUND OF THE INVENTION

[0003] Increased cell proliferation and loss of differentiation havelong been recognized as two major factors that promote neoplastictransformation and progression. This understanding has contributedtremendously to the development of antineoplastic treatments aimed atsuppressing cell proliferation. Antineoplastic therapies, includingradiation and chemotherapeutic agents, ultimately eliminate tumor cellsby the induction of apoptosis, a physiological process of celldestruction. Accumulating evidence suggests that expression of oncogenessensitizes many primary tumors to apoptotic cell death as compared totheir normal counterparts, which provides a critical therapeutic windowfor treatment. However, this therapeutic opportunity is eroded byantiapoptotic mechanisms that are conferred by mutations accumulatedduring the transformation process or induced upon treatment.

[0004] In metazoans oncogenic alterations often sensitize cells toapoptotic stimuli. For example, expression of oncogenic c-myc or theadenovirus early region 1A can increase cellular susceptibility toapoptosis in circumstances such as growth factor deprivation. Thisoncogenic-induced sensitization serves as a physiological barrieragainst tumor development by limiting the expansion of affected cells.Such sensitization to apoptosis by oncogenes also provides a therapeuticwindow for treating many tumors with anticancer agents. However, it iswidely believed that host tumor surveillance mechanisms select forupregulation of antiapoptetic mechanisms during the process oftransformation decreasing the therapeutic benefit of conventionalanti-cancer drugs. Accordingly, by inhibiting critical antiapoptoticmechanisms, sensitivity of tumor cells to therapy-induced apoptosis maybe restored.

[0005] 14-3-3 proteins are dimeric, phosphoserine-binding molecules thatinteract with a number of phospho-proteins involved in controlling celldeath and proliferation. 14-3-3 polypeptides supports cell survival byinhibiting the death promoting activity of its associated proapoptoticpartners. One prominent target of the 14-3-3 polypeptide is Bad. Bad isa proapoptotic member of the Bcl-2 family of apoptosis regulators.Interestingly, Bad is phosphorylated by activated Akt and other kinaseswhich generates a 14-3-3 recognition site, leading to Bad/14-3-3 complexformation. Thus, the phosphorylation of Bad couples multiple survivalsignaling pathways to the cell death machinery. Bad is not the only14-3-3 target with death-promoting activity. ASK1, a Ser/Thr kinase, isa critical element of a death signaling pathway initiated by TNFα, Fasactivation, and the chemotherapeutic agents paclitaxel and cisplatin.14-3-3 binding suppresses the death-promoting activity of ASK1.

[0006] Inhibition of Bad-, ASK-, and FKHRL1-induced apoptosis by 14-3-3raises the possibility that 14-3-3 functions as an antiapoptotic factor(Brunet et al. (1999) Cell 96:857-868; Zha et al. (1996) Cell87:619-628; and Zhang et al. (1999) Proc. Natl. Acad. Sci. 96:8511-8515,all of which are herein incorporated by reference). Because 14-3-3interacts with a large array of ligands involved in both cell death andcell survival, 14-3-3 may be part of a general antiapoptotic mechanismessential for cell survival. 14-3-3 could support cell survival both bysuppressing the activity of proapoptotic proteins and by promoting theactivity of antiapoptotic proteins.

[0007] Given the shortcomings of current chemotherapy and irradiation,namely the lack of response and resistance or tolerance to the variousantineoplastic agents, there is a need for developing additional formsof treatment that can enhance a response of a neoplastic cell to theantineoplastic therapies. The present invention provides a novel methodof treating a neoplastic disorder by modulating the activity of a 14-3-3polypeptide.

SUMMARY OF THE INVENTION

[0008] Methods and compositions for enhancing the death of a neoplasticcell by modulating the activity of a 14-3-3 polypeptide are provided.More specifically, the present invention provides methods andcompositions for enhancing the therapeutic effectiveness of anantineoplastic therapeutic agent.

[0009] In particular, the present invention provides a method foridentifying an agent that selectively inhibits an interaction between a14-3-3 polypeptide and a 14-3-3 ligand. The method comprises (a)contacting a 14-3-3 polypeptide with a 14-3-3 antagonist underconditions that permit formation of a 14-3-3/antagonist complex; (b)contacting the 14-3-3/antagonist complex with a candidate agent; and,(c) determining if the candidate agent disrupts the 14-3-3/antagonistcomplex. The 14-3-3 antagonist used in the methods of the inventioncomprises a polypeptide having an amino acid sequence of SEQ ID NO:1, 2,or a biologically active variants thereof.

[0010] In other embodiments of the present invention, the method ofidentifying an agent that selectively inhibits an interaction between a14-3-3 polypeptide and a 14-3-3 ligand comprises (a) contacting a 14-3-3polypeptide with a candidate agent under conditions that allow for a14-3-3/candidate agent complex to form; (b) contacting said14-3-3/candidate agent complex with a 14-3-3 antagonist; and (c)determining if the 14-3-3 antagonist disrupts the 14-3-3/candidate agentcomplex.

[0011] The present invention further comprises a pharmaceuticalcomposition comprising a therapeutically effective amount of a 14-3-3antagonist and at least one antineoplastic therapeutic agent. In oneembodiment, the 14-3-3 antagonist comprises a polypeptide having anamino acid sequence of SEQ ID NO:1, 2, or a biologically active variantthereof. In other embodiments, the 14-3-3 antagonist comprises an agentidentified by the methods of the present invention.

[0012] The invention further provides a method of enhancing the death ofa neoplastic cell comprising providing to the neoplastic cell atherapeutically effective amount of a 14-3-3 antagonist and at least oneantineoplastic therapeutic agent. The methods of the invention findfurther use in the treatment of neoplastic disorders by theadministration of a therapeutically effective amount of a 14-3-3antagonist and an antineoplastic therapeutic agent to a mammal in needthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 demonstrates the inhibitory effect that the 14-3-3antagonist R18 has on ASK1/14-3-3 interactions. The percentage of ASK1bound to 14-3-3 relative to peptide-free samples is plotted againstincreasing concentrations of the test peptides.

[0014]FIG. 2 demonstrates that the 14-3-3 antagonist R18 competes with aBad derived peptide for 14-3-3 binding.

[0015]FIG. 3 demonstrates that the 14-3-3 antagonist difopein can bind14-3-3 in vivo and can disrupt the interaction of the 14-3-3 polypeptidewith endogenous Raf-1.

[0016]FIG. 4 demonstrates that the 14-3-3 antagonist R18 can induce celldeath in a dose dependent fashion.

[0017]FIG. 5 demonstrates that the cell death induced by the 14-3-3antagonist R18 is dependant on the acidic residues that interact withthe 14-3-3 polypeptide.

[0018]FIG. 6 shows the 14-3-3 antagonist difopein is distributed throughout the cell.

[0019]FIG. 7 demonstrates that diamerization of R 18 enhances itsability to kill cells.

[0020]FIG. 8 demonstrates that the 14-3-3 antagonist difopein can induceapoptotic cell death.

[0021]FIG. 9 demonstrates that the 14-3-3 antagonist difopein killscells through an Akt independent pathway.

[0022]FIG. 10 demonstrates that difopein induced cell death is caspasedependant.

[0023]FIG. 11 demonstrates that the inhibition of 14-3-3polypeptide/ligand interactions can enhance the ability ofchemotherapeutic agents to kill cells.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is directed to the treatment of aneoplastic disorder. Specifically, the present invention provides amethod for enhancing the death of a neoplastic cell comprising theadministration of a therapeutically effective concentration of a 14-3-3antagonist and at least one antineoplastic therapeutic agent. Themethods find use in enhancing the death of a neoplastic cell as comparedto administration of either agent alone. The methods of the presentinvention find use in overcoming the resistance of a cancer to a singletherapeutic agent. The methods of the invention may further allow alower effective dose of the antineoplastic therapeutic agent and therebyreduce the undesirable side effects associated therewith. Hence, themethods of the invention find use in improving the clinical outcome of amammal having a neoplastic disorder comprising administration of anantineoplastic therapeutic agent in combination with a 14-3-3antagonist.

[0025] By “14-3-3 polypeptide” is intended a member of the 14-3-3protein family. 14-3-3 is a family of highly homologous proteins encodedby separate genes. There are seven known mammalian 14-3-3 isoforms,named with Greek letters (β, ε, γ, η, σ, τ, ζ) after their elutionprofile on reversed phase high-performance liquid chromatography(Ichimura et al. (1988) Proc. Natl. Acad. Sci. USA 85:7084-7088 andMartin et al. (1993) FEBS Lett. 331:296-303). The species initiallydesignated α and δ are actually the phosphorylated forms of β and ζ(Aitken et al. (1995) J. Biol. Chem. 270:5706-5709). The 14-3-3 proteinsexist mainly as dimers with a monomeric molecular mass of approximately30,000 and an acidic isoelectric point of 4-5. General properties of the14-3-3 polypeptides can further be found in Fu et al. (2000) Annu. Rev.Pharmacol. Toxicol. 40:617-647, herein incorporated by reference. Thenucleic acid and amino acid sequences of various 14-3-3 family memberscan be found in, for example, Leffer et al. (1993) J. Mol. Biol.231:982-998, which is herein incorporated by reference. Homologues of14-3-3 proteins have also been found in a broad range of eukaryoticorganisms. As discussed herein, the 14-3-3 polypeptides have a varietyof biological functions. By “modulation of 14-3-3” activity is intendedany alteration of the 14-3-3 polypeptide activity that results indisrupting the ability of the 14-3-3 polypeptide to interact with a14-3-3 ligand, particularly a ligand comprising a proapoptoticpolypeptide. Disruption of the 14-3-3 polypeptide/ligand interaction ischaracterized in vivo by enhanced cell death and/or the sensitization ofthe cell to antineoplastic therapeutic agents. In addition, thedisruption of the 14-3-3 polypeptide/ligand interaction can further becharacterized by alterations in cell differentiation, cellproliferation, cell cycle regulation, viral transformation, bacterialpathogenesis, or apoptosis. Both in vivo and in vitro assays that can beused to assay for these interactions are discussed more fully below.

[0026] As defined herein, a “14-3-3 antagonist” comprises a chemicalcompound, a mixture of chemical compounds, or a biological macromoleculethat modulates the activity of the 14-3-3 polypeptide. The 14-3-3antagonist of the invention can modulate the activity of the 14-3-3polypeptide by interacting directly with the 14-3-3 polypeptide. Inspecific embodiments, the direct interaction of the 14-3-3 antagonistwith the 14-3-3 polypeptide disrupts the interaction of the 14-3-3polypeptide with a 14-3-3 ligand. For instance, the 14-3-3 antagonistcan directly disrupt the interaction of the 14-3-3 polypeptide with adeath-regulatory protein including, for example, Bad (Zha et al. (1996)Cell 87:619-628); ASK1 (Zhang et al. (1999) Proc. Natl. Acad. Sci.96:8511-8515); p53; PI 3-kinase (Bonnefoy et al. (1995) Proc. Natl.Acad. Sci. 92:10142-10146); Bcr-Abl (Reuther et al. (1994) Science266:129-133); IGF-IR (Craparo et al. (1997) J. Biol. Chem.272:11663-11669); and Raf-1 (Fantl et al. (1994) Nature 371:612-614;Freed et al. (1994) Science 265:533-535; and Fu et al. (1994) Science266:126-129). As discussed in more detail below, the 14-3-3 antagonistcan comprise the amino acid sequence set forth in SEQ ID NO:1, 2 orbiologically active variants thereof. In other embodiments the 14-3-3antagonist comprises an agent identified by the methods described below.

[0027] The 14-3-3 polypeptide is dimeric and comprises two amphipathicpeptide-binding grooves that are arranged in an antiparallel fashion(Liu et al. (1995) Nature 376:191-194 and Petosa et al. (1998) J. Biol.Chem. 273:16305-16310, both of which are herein incorporated byreference). The inner surface of the groove is formed by the fourhelices (α3, α5, α7 and α9) and is characterized as having a cluster ofbasic and polar residues (from α3 and α5) on one side and a cluster ofhydrophobic residues (from α7 and α9) on the other side. The amphipathicgroove may bind different peptide motifs and induce either homodimer orheterodimer formation in its target proteins (i.e., 14-3-3 ligands).Residues of the amphipathic groove of bovine 14-3-3 ζ include N38, E39,R41, N42, L43, S45, V46, K49, N50, V52, G53, R56, S57, R60, E113, F117,K120, M121, D124, Y125, R127, Y128, P165, I166, G169, L172, N173, V176,Y179, E180, D213, L216, I217, L220, D223, N224, L227, W228.

[0028] As used herein, a “14-3-3 amphipathic groove binding antagonist”interacts with the 14-3-3 polypeptide with at least one of the aminoacids comprising the conserved amphipathic groove of the 14-3-3polypeptide dimer and thereby prevents the association of 14-3-3 ligandsthat interact at the amphipathic groove. See, for example, Petosa et al.(1998) The Journal of Biological Chemistry 273:16305-16310, hereinincorporated by reference, for the crystal structure of the amphipathicgroove. Petosa et al. further show that the 14-3-3 antagonist providedin SEQ ID NO:1 is a 14-3-3 amphipathic groove binding antagonist. The14-3-3 antagonist set forth in SEQ ID NO:2 is also an amphipathic groovebinding antagonist. As demonstrated elsewhere herein, the binding of the14-3-3 antagonist of SEQ ID NO:2 (difopein) inhibits the interaction ofthe 14-3-3 polypeptide with Raf-1, a prototypical 14-3-3 amphipathicgroove binding protein.

[0029] It is further recognized that the 14-3-3 antagonist used in themethods of the present invention may indirectly modulate the activity ofthe 14-3-3 polypeptide. An indirect interaction encompasses, forexample, an alteration in the activity of a polypeptide that influencesthe ability of the 14-3-3 polypeptide to interact with a 14-3-3 ligand(i.e., a proapoptotic protein). For instance, the interaction of the14-3-3 polypeptide with many of the ligands is regulated by specificphosphorylation of the 14-3-3 ligand. The activities of the kinases thatphosphorylate these ligands can therefore regulate 14-3-3polypeptide/ligand interactions. See, for example, Muslin et al. (1996)Cell 84:889-897; Yaffe et al. (1997) Cell 91:961-971; and Furakawa etal. (1993) Biochem. Biophys. Res. Commun. 194:144-149, all of which areherein incorporated by reference. Hence, in specific embodiments of thepresent invention, the 14-3-3 antagonist can act indirectly on the14-3-3 polypeptide by influencing regulatory factors, such as kinasesthat regulate the phosphorylation state of the various 14-3-3 ligands.

[0030] Administration of the 14-3-3 antagonist to a cell enhances celldeath. By “enhance” is intended any increase in the level of cell deathupon administration of the 14-3-3 antagonist when compared to the extentof cell death occurring in the absence of the 14-3-3 antagonist.Administration of the 14-3-3 antagonist results in the enhancedsusceptibility or sensitization of a neoplastic cell to respond to anantineoplastic therapeutic agent. By “sensitization” is intended thecombined administration of the antineoplastic therapeutic agent and the14-3-3 antagonist produces an anti-cancer effect (i.e., prohibition ofcellular proliferation or potentiation of cell death) which exceeds thetherapeutic effect of either the 14-3-3 antagonist and theantineoplastic therapeutic agent alone.

[0031] The present invention therefore provides a method to treat aneoplastic disease or disorder. By “treatment or prevention” is intendedthe alleviation of the signs, symptoms, or causes of a disease, or anyother desired alteration of a biological system. Accordingly, the methodof the invention “prevents” (i.e., delays or inhibits) and/or “reduces”(i.e., decrease, slows, or ameliorates) the detrimental effects of theneoplastic disease or disorder in the mammal receiving the therapy.

[0032] As used herein, a “neoplastic disease or disorder” ischaracterized by one or more of the following properties: cell growth isnot regulated by the normal biochemical and physical influences in theenvironment; anaplasia (i.e., lack of normal coordinated celldifferentiation); and in some instances, metastasis. The term cancer,neoplasia, and malignancy are used interchangeably herein. Neoplasticdiseases include, for example, anal carcinoma, bladder carcinoma, breastcarcinoma, cervix carcinoma, chronic lymphocytic leukemia, chronicmyelogenous leukemia, endometrial carcinoma, hairy cell leukemia, headand neck carcinoma, lung (small cell) carcinoma, multiple myeloma,non-Hodgkin's lymphoma, follicular lymphoma, ovarian carcinoma, braintumors, colorectal carcinoma, hepatocellular carcinoma, Kaposi'ssarcoma, lung (non-small cell carcinoma), melanoma, pancreaticcarcinoma, prostate carcinoma, renal cell carcinoma, and soft tissuesarcoma. Additional neoplastic disorders can be found in, for example,Isselbacher et al. (1994) Harrison's Principles oflnternal Medicine1814-1877, herein incorporated by reference.

[0033] Any antineoplastic agent (i.e., chemotherapeutic, radiation, orbiological response modifiers) can be used in the methods of the presentinvention. It is understood that the antineoplastic agent may affectneoplastic cells by a variety of mechanisms, including killing ordecreasing viability, by apoptosis or various other cellular mechanisms.Regardless of the mechanism of the antineoplastic agent, the 14-3-3antagonist will sensitize the neoplastic cell to the antineoplasticagent. In any particular embodiment of the invention, the antineoplastictherapeutic agent will be selected with reference to factors such as thetype of neoplastic disorder and the efficacy of the antineoplastic agentfor treating the desired neoplastic disorder.

[0034] Chemotherapeutic agents include, but are not limited to,Aminoglutethimide; Asparaginase; Bleomycin; Busulfan; Carboplatin;Carmustine (BCNU); Chlorambucil; Cisplatin (cis-DDP); Cyclophosphamide;Cytarabine HCl; Dacarbazine; Dactinomycin; Daunorubicin HCl; DoxorubicinHCl; Estramustine phosphate sodium; Etoposide (VP-16); Floxuridine;Fluorouracil (5-FU); Flutamide; Hydroxyurea (hydroxycarbamide);Ifosfamide; Interferon α-2a, α-2b, Lueprolide acetate (LHRH-releasingfactor analogue); Lomustine (CCNU); Mechlorethamine HCl (nitrogenmustard); Melphalan; Mercaptopurine; Mesna; Methotrexate (MTX);Mitomycin; Mitotane (o.p'-DDD); Mitoxantrone HCl; Octreotide;Paclitaxel; Plicamycin; Procarbazine HCl; Streptozocin; Tamoxifencitrate; Thioguanine; Thiotepa; Vinblastine sulfate; Vincristinesulfate; Amsacrine (m-AMSA); Azacitidine; Hexamethylmelamine (HMM);Interleukin 2; Mitoguazone (methyl-GAG; methyl glyoxalbis-guanylhydrazone; MGBG); Pentostatin; Semustine (methyl-CCNU);Teniposide (VM-26); paclitaxel and other taxanes; and Vindesine sulfate.

[0035] Current methods for the treatment of many neoplastic disorderscomprise the use of multiple anti-neoplastic therapeutic agents (i.e.,polytherapy). Such polytherapies are known in the art and include, butare not limited to, the combined administration of cyclophosphamide,methotrexate, and 5-fluorouracil for breast cancer; cyclophosphamide,doxorubicin, methotrexate, and procarbazine for non-small cell lungcancer; 5-fluorouracil and levamisole for colon cancer; and,cyclophosphamide, doxorubicin, vincristine, and prednisone fornon-Hodgkin's lymphoma. Additional examples of polytherapy regimesuseful in the methods of the invention can be found in, for example,DiPiro et al. (1993) Pharmacotherapy: A Pathophysiological Approach,2^(nd) ed., Appleton and Lange, Norwalk, Conn.

[0036] Additional antineoplastic therapeutic agents which find use inthe methods of the present invention include biological responsemodifiers. As used herein “biological response modifiers” comprise anyagent that functions by altering the host response to cancer, ratherthan by direct cytotoxicity. Biological response modifiers include, forexample, monoclonal antibodies and cytokines. See, for example,Isselbacher et al. (1994) Harrison's Principles of Internal Medicine,1834-1841, which is herein incorporated by reference. Cytokines are agroup of intercellular messenger proteins that are key immunoregulatorycompounds. They comprise the largest group of biologic therapeutics inclinical trials and include interferons (i.e., Type I interferons suchas INF-α and INF-β and Type II interferons such as INF-γ), interleukins,and hematopoeitic growth factors (i.e., erythropoietin,granulocyte-macrophage colony stimulating factor (GM-CSF) andgranulocyte colony stimulating factor (G-CSF)).

[0037] As used herein “radiation” is intended to include any treatmentof a neoplastic cell or subject by photons, neutrons, electrons, orother type of ionizing radiation. Such radiations include, but are notlimited to, X-ray, gamma-radiation, or heavy ion particles, such asalpha or beta particles. Additionally, the radiation may be radioactive.The means for irradiating neoplastic cells in a subject are well knownin the art and include, for example, external beam therapy, andbrachytherapy.

[0038] 14-3-3 Antagonists

[0039] The present invention provides 14-3-3 antagonists that can eitherbe used alone or in combination with an antineoplastic agent to enhancecell death of a neoplastic cell. The 14-3-3 antagonists useful in themethods of the present invention can comprise a polypeptide having theamino acid sequence of SEQ ID NOS:1 or 2. The polypeptide of SEQ ID NO:1is a 14-3-3 amphipathic groove binding antagonist referred to herein as“R18”. Petosa et al. have demonstrated that the R18 peptide binds 14-3-3polypeptide within the conserved groove and inhibits the interaction ofvarious 14-3-3 ligands, including for example, Raf-1 kinase andexoenzyme S. ((1998)J. Biol. Chem. 273:16305-16310).

[0040] Another 14-3-3 antagonist of the present invention comprises thepolypeptide of SEQ ID NO:2 and is referred to herein as “difopein”. Thedifopein 14-3-3 antagonist comprises a 62 amino acid polypeptide thatcomprises two R18 monomers (a.a. 6-25 and 37-56 of SEQ ID NO:2)separated by a non-repeat linker sequence (a.a. 26-36 of SEQ ID NO:2).The 14-3-3 antagonists set forth in SEQ ID NOS:1 and 2 are characterizedby their ability to modulate the activity of the 14-3-3 polypeptide(i.e., disrupt 14-3-3 polypeptide/ligand interactions and/or enhancecell death).

[0041] Biologically active variants of 14-3-3 antagonists set forth inSEQ ID NOS:1 and 2 are also encompassed by the methods of the presentinvention. Such variants should retain the biological activity of the14-3-3 antagonist (i.e., the ability to modulate 14-3-3 activity andthereby disrupt the 14-3-3 polypeptide/ligand interactions, enhance celldeath, and/or sensitize the neoplastic cell to an antineoplastictherapeutic agent). Such activity may be measured using standardbioassays. Representative assays detecting the activity of the 14-3-3antagonists include, for example, various in vitro binding assays asdescribed in Wang et al. (1999) Biochem. 38:12499-12504; Petosa et al.(1998) J. Biol. Chem. 273:16305-16310; and Wang et al. (1998)J. Biol.Chem. 273:16297-16304, all of which are herein incorporated byreference. Further assays that measure cell death include, for example,visual inspection for the morphological signs of cell death, an increasein DNA fragmentation, and an increased activity of polypeptides involvedin apoptosis. See, for example, the methods described herein and Zhanget al. (1999) Proc. Natl. Acad. Sci. USA 96:8511-8515 and U.S. Pat. No.5,821,072, all of which are herein incorporated by reference.

[0042] Suitable biologically active variants can be fragments,analogues, and derivatives of the 14-3-3 antagonists set forth in SEQ IDNOS:1 and 2. By “fragment” is intended a polypeptide consisting of onlya part of the intact 14-3-3 antagonist polypeptide sequence. Thefragment can be a C-terminal deletion or N-terminal deletion of the14-3-3 antagonist polypeptide. By “analogue” is intended an analogue ofeither the full length polypeptide having biological activity or afragment thereof, that includes a native sequence and structure havingone or more amino acid substitutions, insertions, or deletions. By“derivative” is intended any suitable modification of the 14-3-3antagonist polypeptide or fragments thereof, or their respectiveanalogues, such as glycosylation, phosphorylation, or other addition offoreign moieties, so long as the activity is retained.

[0043] Preferably, naturally or non-naturally occurring variants of a14-3-3 polypeptide antagonist have amino acid sequences that are atleast 70%, preferably 80%, more preferably, 85%, 90%, 91%, 92%, 93%, 94%or 95% identical to the amino acid sequence to the amino acid sequenceof SEQ ID NOS:1 or 2 or to a shorter portion of these molecules. Morepreferably, the molecules are 96%, 97%, 98% or 99% identical. Percentsequence identity is determined using the Smith-Waterman homology searchalgorithm using an affine gap search with a gap open penalty of 12 and agap extension penalty of 2, BLOSUM matrix of 62. The Smith-Watermanhomology search algorithm is taught in Smith and Waterman, Adv. Appl.Math. (1981) 2:482-489. A variant may, for example, differ by as few as1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3,2, or even 1 amino acid residue.

[0044] With respect to optimal alignment of two amino acid sequences,the contiguous segment of the variant amino acid sequence may haveadditional amino acid residues or deleted amino acid residues withrespect to the reference amino acid sequence. The contiguous segmentused for comparison to the reference amino acid sequence will include atleast 10 contiguous amino acid residues, and may be 12, 13, 14, 15, 16,17, 18, 20, 25, 30 or more amino acid residues. Corrections for sequenceidentity associated with conservative residue substitutions or gaps canbe made (see Smith-Waterman homology search algorithm).

[0045] The art provides substantial guidance regarding the preparationand use of such variants, as discussed further below. A fragment of a14-3-3 polypeptide antagonist will generally include at least about 10contiguous amino acid residues of the full-length molecule, preferablyabout 15-25 contiguous amino acid residues of the full-length 14-3-3antagonist.

[0046] For example, conservative amino acid substitutions may be made atone or more predicted, preferably nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe sequence of SEQ ID NOS:1 and 2 without altering its biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Such substitutions would not be made for conserved aminoacid residues, or for amino acid residues residing within a conservedmotif.

[0047] Alternatively, variant 14-3-3 antagonist nucleotide sequences canbe made by introducing mutations randomly along all or part of a 14-3-3antagonist sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for 14-3-3 antagonist activity toidentify mutants that retain activity. Following mutagenesis, theencoded polypeptide can be expressed recombinantly, and the activity ofthe polypeptide can be determined using standard assay techniquesdescribed herein.

[0048] Alternatively, the 14-3-3 antagonist can be synthesizedchemically, by any of several techniques that are known to those skilledin the peptide art. See, for example, Li et al. (1983) Proc. Natl. Acad.Sci. USA 80:2216-2220, Steward and Young (1984) Solid Phase PeptideSynthesis (Pierce Chemical Company, Rockford, Ill.), and Baraney andMerrifield (1980) The Peptides: Analysis, Synthesis, Biology, ed. Grossand Meinhofer, Vol. 2 (Academic Press, New York, 1980), pp. 3-254,discussing solid-phase peptide synthesis techniques; and Bodansky (1984)Principles of Peptide Synthesis (Springer-Verlag, Berlin) and Gross andMeinhofer, eds. (1980) The Peptides: Analysis, Synthesis, Biology, Vol.1 (Academic Press, New York), discussing classical solution synthesis.The 14-3-3 antagonist can also be chemically prepared by the method ofsimultaneous multiple peptide synthesis. See, for example, Houghten(1984) Proc. Natl. Acad. Sci. USA 82:5131-5135; and U.S. Pat. No.4,631,211.

[0049] It is further recognized that various other 14-3-3 antagonistsknown in the art can be used in the methods of the present invention tosensitize a neoplastic cell to an antineoplastic therapeutic agent. Such14-3-3 antagonists include phosphoserine ligands, such as Raf-1 kinaseand Bad which are characterized as having a phosphorylated consensusmotif comprising Arg-Ser-Xaa-pSer-Xaa-Pro (SEQ ID NO:3), where ‘xaa’represents any residue and pSer is a phosphoserine residue. Examples ofsuch 14-3-3 antagonist are disclosed in U.S. Pat. Nos. 5,948,765;5,856,445; and 5,597,719; all of which are herein incorporated byreference. Other 14-3-3 antagonists of interest include, for example,IGF-1 receptor (Craparo et al. (1997) J. Biol. Chem. 272:11663-11669),IRSI (Craparo et al. (1997) J. Biol. Chem. 272:1663-1669 and Ogihara etal. (1997) J. Biol. Chem. 272:25267-25274), the 43 KDa inositolpolyphosphate 5-phosphatase (5-phosphatase) (Campbell et al. (1997)Biochemistry 36:15363-15370), the glycoprotein Ibα and the exoenzyme S(ExoS) from Pseudomonas aeruginosa (Lana et al. (2000) Biochem. J.349:697-701 and Masters et al. (1999) Biochemistry 38:5216-5221). All ofthese references are herein incorporated by reference.

[0050] In addition to peptides consisting only of naturally-occurringamino acids, peptidomimetics or peptide analogs are also provided.Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepolypeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics” (Luthman et al. (1996) A Textbook of DrugDesign and Development, 14:386-406, 2nd Ed., Harwood AcademicPublishers; Joachim Grante et al. (1994) Chem. Int. Ed. Engl.33:1699-1720; Fauchere et al. (1986) Adv. Drug Res. 15:29; Veber et al.(1985) TINS, 392; and Evans et al. (1987) J. Med. Chem. 30:1229, all ofwhich are incorporated herein by reference). Peptide mimetics that arestructurally similar to therapeutically useful polypeptides may be usedto produce an equivalent or enhanced therapeutic or prophylactic effect.Accordingly, additional 14-3-3 antagonists can be identified usingtechniques known in the art for the development of peptide mimics.

[0051] Briefly, these methods involve the identification andcharacterization of the 14-3-3 polypeptide as well as the 14-3-3 ligandusing X-ray crystallography, NMR, or other structure analysis methodssuch as the DOCK program (Kuntz et al. (1982) J. Mol. Biol. 161:269 andKuntz (1992) Science 257:1078) or variants thereof. Potentialtherapeutic drugs may be designed rationally on the basis on structuralinformation provided thereby. The X-ray crystal structure for the 14-3-3amphipathic groove binding antagonist, R18 (SEQ ID NO:1), is known(Petosa et al. (1998) The Journal of Biological Chemistry273:16305-16310. Accordingly, a pharmacophore hypothesis can bedeveloped, and compounds subsequently made and tested in an assay systemto determine if they are effective 14-3-3 antagonists. The assay systemused to characterize the potential 14-3-3 antagonist candidate may bebased upon the displacement of a 14-3-3 ligand from the 14-3-3polypeptide as described elsewhere herein.

[0052] Methods of Identifying a 14-3-3 Antagonist

[0053] The present invention provides 14-3-3 antagonists comprising theamino acid sequence of SEQ ID NOS:1 and 2. As demonstrated herein, R18and difopein can be used alone or in combination with an antineoplastictherapeutic agent for the treatment of a neoplastic disorder. Thesefindings indicate that 14-3-3 antagonists find use as therapeutic agentsin the treatment of neoplastic disorders. Accordingly, the presentinvention provides methods for identifying additional 14-3-3antagonists. Specifically, the present invention provides a method foridentifying an agent that selectively inhibits an interaction between a14-3-3 polypeptide and a 14-3-3 ligand (i.e., a proapoptoticpolypeptide).

[0054] In one embodiment, the method comprises (a) contacting a 14-3-3polypeptide with a 14-3-3 antagonist under conditions that permitformation of a 14-3-3/antagonist complex; (b) contacting the14-3-3/antagonist complex with a candidate agent; and, (c) determiningif the candidate agent disrupts the 14-3-3/antagonist complex. Adecrease in binding in the presence of the candidate agent compared tothat in the absence of a candidate agent indicates that the agentinhibits the direct binding of the 14-3-3 antagonist to the 14-3-3polypeptide. In this manner, the ability of the candidate agent tocompete with the 14-3-3 antagonist for binding is measured. The 14-3-3antagonist used in the methods of the invention comprises a polypeptidehaving an amino acid sequence of SEQ ID NO:1, 2, or a biologicallyactive variant thereof.

[0055] In another embodiment, the candidate agent is allowed to interactwith the 14-3-3 polypeptide. The 14-3-3 polypeptide/candidate agent issubsequently contacted with the 14-3-3 antagonist of SEQ ID NO:1, 2, ora biologically active variant thereof. The ability of the 14-3-3antagonist to disrupt the 14-3-3 polypeptide/candidate agent is assayed.

[0056] Both in vivo and in vitro competitive binding assays are known inthe art and can be adapted for use in the methods of the presentinvention. In vivo assays for 14-3-3 interaction include, for example,the yeast two-hybrid system or a fluorescence resonant energy transferbased assay. In vitro assays include, for example, ELISA/solid phasebinding assays, fluorescence polarization based assays, and fluorescenceresonant energy transfer assays.

[0057] In fluorescence polarization assays, the 14-3-3 antagonist (i.e.,SEQ ID NO:1 or 2) is conjugated to a small molecule fluorophore, i.e.fluorescein or Oregon green. Binding of the tagged 14-3-3 antagonist topurified 14-3-3 would cause a decrease in the mobility of the 14-3-3antagonist and thus, increase the polarization of the emitted light fromthe fluorophore. This technique thereby allows for the identification ofnovel 14-3-3 antagonists.

[0058] Fluorescence resonant energy transfer uses two fluorescentlytagged species, where the emission spectrum of the shorter wavelengthtag overlaps the excitation spectrum of the longer wavelength tag. Closeproximity of the two molecules induced by binding allows nonradiativeexcitation of the long wavelength tag when the short wavelength tag isexcited. In this method, two DNA constructs coding for the 14-3-3polypeptide and 14-3-3 antagonist are tagged with ECFP(cyan) andEYFP(yellow). Upon expression in vivo, energy transfer in the celllysates can be observed. It is recognized that such assays can beadapted to an in vitro format.

[0059] It is further recognized that each of these methods can beadapted for high-throughput screening methods using the 14-3-3antagonists of the present invention to identify new 14-3-3 antagonists.Hence, the methods of the present invention allow for thehigh-throughput screening of various candidate agents to identifypotentially therapeutically valuable 14-3-3 antagonists. A candidateagent may be a chemical compound, a mixture of chemical compounds, or abiological macromolecule. Such candidate agents can be contained invarious agent banks including, for example, compound libraries, peptidelibraries, and the like.

[0060] Methods of Administration

[0061] Delivery of a therapeutically effective amount of a 14-3-3antagonist may be obtained via administration of a pharmaceuticalcomposition comprising a therapeutically effective dose of this agent.By “therapeutically effective amount” or “dose” is meant theconcentration of a 14-3-3 antagonist that is sufficient to elicit thedesired therapeutic effect, i.e., the death of a neoplastic cell.

[0062] A therapeutically effective amount of a 14-3-3 antagonist whenused in combination with an antineoplastic therapeutic agent issufficient to enhance the death of a neoplastic cell. Accordingly, inthis embodiment, an effective amount of the 14-3-3 antagonist augmentsthe clinical outcome of a traditional antineoplastic therapeutic agent.As such, a therapeutically effective dose can be assayed via thesensitization of a neoplastic cell to the antineoplastic therapeuticagent; a decrease in tumor volume; or an increase in cell death. Hence,a therapeutically effective amount is characterized by an improvement inclinical symptoms of a neoplastic disorder and/or a reduction in theeffective dosage concentration of the antineoplastic therapeutic agent.

[0063] Methods to determine if the neoplastic disorder has been treatedare well known to those skilled in the art and include, for example, adecrease in the number of neoplastic cells (ie., a decrease in cellproliferation or a decrease in tumor size). It is recognized that thetreatment of the present invention may be a lasting and completeresponse or can encompass a partial or transient clinical response. Seefor example, Isselbacher et al. (1996) Harrison's Principles of InternalMedicine 13 ed., 1814-1882, herein incorporated by reference.

[0064] Assays to test for the sensitization or the enhanced death ofneoplastic cells are well known in the art, including, for example,standard dose response assays that assess cell viability; agarose gelelectrophoresis of DNA extractions or flow cytometry to determine DNAfragmentation, a characteristic of cell death; assays that measure theactivity of polypeptides involved in apoptosis; and assay formorphological signs of cell death. The details regarding such assays aredescribed elsewhere herein. Other assays include, chromatin assays(i.e., counting the frequency of condensed nuclear chromatin) or drugresistance assays as described in, for example, Lowe et al. (1993) Cell74:957-697, herein incorporated by reference. See also U.S. Pat. No.5,821,072, also herein incorporated by reference.

[0065] In addition, assays to test for the sensitization of a neoplasticcell and thereby determine a therapeutically effective dose of a 14-3-3antagonist alone, or in combination with an antineoplastic therapeuticagent, can be preliminarily evaluated by using a tumor growth regressionassay which assesses the ability of tested compounds to inhibit thegrowth of established solid tumors in mice. The assay can be performedby implanting tumor cells into the fat pads of nude mice. Tumor cellsare then allowed to grow to a certain size before the agents areadministered. The volumes of tumors are monitored for a set number ofweeks, e.g., three weeks. General health of the tested animals is alsomonitored during the course of the assay.

[0066] It is contemplated that the 14-3-3 antagonists of the presentinvention will be administered to a subject (i.e., a mammal, preferablya human) in therapeutically effective amounts. That is, in an amountsufficient to enhance the death of a neoplastic cell. The effectiveamount of the 14-3-3 antagonist composition will vary according to theweight, sex, age, and medical history of the subject. Other factorswhich influence the effective amount may include, but are not limitedto, the severity of the patient's condition, the neoplastic disorderbeing treated, the stability of the 14-3-3 antagonist, and, if desired,the antineoplastic therapeutic agent being administered with the 14-3-3antagonist. Typically, for a human subject, an effective amount willrange from about 0.001 mg/kg of body weight to about 30 mg/kg of bodyweight. A larger total dose can be delivered by multiple administrationsof the agent. Methods to determine efficacy and dosage are known tothose skilled in the art. See, for example, Isselbacher et al. (1996)Harrison's Principles of Internal Medicine 13 ed., 1814-1882, hereinincorporated by reference.

[0067] In another embodiment of the invention, the pharmaceuticalcomposition comprising the therapeutically effective dose of a 14-3-3antagonist is administered intermittently. By “intermittentadministration” is intended administration of a therapeuticallyeffective dose of a 14-3-3 antagonist, followed by a time period ofdiscontinuance, which is then followed by another administration of atherapeutically effective dose, and so forth. Administration of thetherapeutically effective dose may be achieved in a continuous manner,as for example with a sustained-release formulation, or it may beachieved according to a desired daily dosage regimen, as for examplewith one, two, three, or more administrations per day. By “time periodof discontinuance” is intended a discontinuing of the continuoussustained-released or daily administration of the 14-3-3 antagonist. Thetime period of discontinuance may be longer or shorter than the periodof continuous sustained-release or daily administration. During the timeperiod of discontinuance, the 14-3-3 antagonist level in the relevanttissue is substantially below the maximum level obtained during thetreatment. The preferred length of the discontinuance period depends onthe concentration of the effective dose and the form of 14-3-3antagonist used. The discontinuance period can be at least 2 days, atleast 4 days or at least 1 week. In other embodiments, the period ofdiscontinuance is at least 1 month, 2 months, 3 months, 4 months orgreater. When a sustained-release formulation is used, thediscontinuance period must be extended to account for the greaterresidence time of the 14-3-3 antagonist at the site of injury.Alternatively, the frequency of administration of the effective dose ofthe sustained-release formulation can be decreased accordingly. Anintermittent schedule of administration of 14-3-3 antagonist cancontinue until the desired therapeutic effect, and ultimately treatmentof the neoplastic disease or disorder is achieved.

[0068] It is recognized that the 14-3-3 antagonist and theantineoplastic therapeutic agent can be administered in a fixedcombination (i.e., a single pharmaceutical formulation that containsboth active materials). Alternatively, the 14-3-3 antagonist may beadministered simultaneously with the antineoplastic therapeutic agent.In another embodiment, the 14-3-3 antagonist and the antineoplastictherapeutic agent are administered sequentially (i.e., administration ofthe 14-3-3 antagonist begins shortly after the end of the antineoplastictherapeutic agent regime or, alternatively, administration of the 14-3-3antagonist precedes the administration of the antineoplastic therapeuticagent). One of skill in the art will recognized that the most preferredmethod of administration will allow the desired therapeutic effect,i.e., the enhanced cell death of a neoplastic cell.

[0069] All conventional forms of administration of the 14-3-3 antagonistand the antineoplastic may be used (i.e., tablets, capsules, dragees,syrups, solutions and suspensions) for the methods of the presentinvention. Preferred forms of systemic administration of thepharmaceutical compositions include injection, typically by intravenousinjection. Other injection routes, such as subcutaneous, intramuscular,or intraperitoneal can also be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrates such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

[0070] The 14-3-3 antagonists of the present invention may be dissolvedin any physiologically tolerated liquid in order to prepare aninjectable bolus. It is generally preferable to prepare such a bolus bydissolving the molecule in normal saline.

[0071] It is further recognized that when the 14-3-3 antagonist ofchoice is a polypeptide, the antagonist can be administered to theneoplastic cell using conventional gene therapy techniques. In suchinstances, the nucleotide sequence encoding the 14-3-3 antagonist isoperably linked to a promoter that is active in the targeted neoplasticcell. The DNA construct containing the 14-3-3 antagonist is subsequentlyinserted into a vector and introduced in the subject.

[0072] Pharmaceutical Compositions

[0073] The present invention also provides pharmaceutical formulationsor compositions, both for veterinary and for human medical use, whichcomprise a 14-3-3 antagonist alone or in combination with one or morepharmaceutically acceptable carriers. In additional embodiments, thepharmaceutical compositions comprising the 14-3-3 antagonist furthercomprise an antineoplastic therapeutic agent. The carrier(s) must bepharmaceutically acceptable in the sense of being compatible with theother ingredients of the formulation and not unduly deleterious to therecipient thereof.

[0074] The 14-3-3 antagonist compositions can be formulated according toknown methods to prepare pharmaceutically useful compositions, such asby admixture with a pharmaceutically acceptable carrier vehicle.Suitable vehicles and their formulation are described, for example, inRemington's Pharmaceutical Sciences (16th ed., Osol, A. (ed.), Mack,Easton Pa. (1980)). A pharmaceutically acceptable composition suitablefor effective administration will contain an effective amount of the14-3-3 antagonist. Additional pharmaceutical methods may be employed tocontrol the duration of action. Controlled release preparations may beachieved by the use of polymers to complex or absorb the 14-3-3antagonist. The controlled delivery may be exercised by selectingappropriate macromolecules (for example, polyesters, polyamino acids,polyvinyl pyrrolidone, ethylene-vinylacetate, hydrogels, poly(lacticacid) methylcellulose, carboxymethylcellulose, or protamine sulfate).Altering the concentration of such macromolecules may also control therate of drug release.

[0075] Alternatively, it is possible to entrap the therapeutic agents inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrylate)microcapsules, respectively, or in a colloid drug delivery system, forexample, liposomes, albumin, microspheres, microemulsions,nanoparticles, nanocapsules, or in macroemulsions. Such teachings aredisclosed in Remington's Pharmaceutical Sciences (1980), hereinincorporated by reference.

[0076] Articles of Manufacture

[0077] The present invention also includes an article of manufactureproviding a 14-3-3 antagonist. The article of manufacture may containthe 14-3-3 antagonist alone or in combination with an antineoplastictherapeutic agent. The article of manufacture can include a vial orother container that contains a composition suitable for the presentmethod together with any carrier, either dried or in liquid form. Thearticle of manufacture further includes instructions in the form of alabel on the container and/or in the form of an insert included in a boxin which the container is packaged, for the carrying out the method ofthe invention. The instructions can also be printed on the box in whichthe vial is packaged. The instructions contain information such assufficient dosage and administration information so as to allow thesubject or a worker in the field to administer the pharmaceuticalcomposition. It is anticipated that a worker in the field encompassesany doctor, nurse, technician, spouse, or other caregiver that mightadminister the composition. The pharmaceutical composition can also beself-administered by the subject.

[0078] Having now generally described this invention, the same will bebetter understood by reference to certain specific examples which areincluded herein for purposes of illustration only, and are not intendedto be limiting of the invention, unless specified.

[0079] Experimental

EXAMPLE 1 R18 is a 14-3-3 Antagonist that Disrupts 14-3-3/LigandInteractions

[0080] To control 14-3-3/ligand interactions, the interaction of the14-3-3 antagonist, R18, with the 14-3-3 polypeptide was characterized.The R18 polypeptide (SEQ ID NO:1) was isolated from a phage displaylibrary based on its affinity for 14-3-3 proteins. It appears that thispeptide binds different isoforms of the 14-3-3 with similar affinity(KD=80 nM), thus it may serve as a general 14-3-3 inhibitor. ImportantlyR18 exhibited high specificity for 14-3-3 proteins. See, for example,Wang et al. (1999) Biochemistry 38:12499-12504.

[0081] To determine the effectiveness of various 14-3-3 antagonist,including R18, S-Raf259, and pS-Raf-259, the peptide antagonists werepreincubated with immobilized His-14-3-3ζ before adding [³⁵S]-labeledASK1. After washing, 14-3-3-bound ASK1 was quantified by PhosphorImager.The percentage of ASK1 bound to 14-3-3 relative to peptide-free samplesis plotted against increasing concentration of the test peptides (FIG.1). R18 has a potent inhibitory affect on the interaction of 14-3-3ζwith ASK1. The phosphorylated peptide pS-Raf-259 that interacts at theamphipathic groove of the 14-3-3 polypeptide was also an effectiveantagonist. However, as shown in FIG. 1, the R18 antagonist has astronger inhibitory affect on 14-3-3ζ/ASK1 than the phosphorylatedpS-Raf-259 polypeptide.

[0082]FIG. 2 demonstrates that the R18 antagonist (SEQ ID NO:1) competeswith a Bad derived peptide for 14-3-3 binding. R18 was covalentlycoupled to Sepharose beads. The beads were then incubated with 100 nM14-3-3ζ dimer along with the indicated concentrations of syntheticphosphopeptides derived from Bad. pS 136 contains a 14-3-3 binding site,while pS 112 is a similar peptide incapable of binding 14-3-3. Unbound14-3-3 was washed away from the beads and the remaining proteins werevisualized by SDS-PAGE and sliver staining. The data demonstrates thatthe R18 polypeptide of SEQ ID NO:1 competes with a Bad derived peptidefor 14-3-3 binding. See also, Masters et al. (2001) MolecularPharmacology 60: 1325-1331, herein incorporated by reference.

[0083] Difopein is a 62 amino acid polypeptide having 14-3-3 antagonistactivity. The amino acid sequence of difopein is shown in SEQ ID NO:2and comprises two R18 monomers (amino acids 6-25 and 37-56 of SEQ IDNO:2) separated by non-repeat linker sequences (amino acids 26-36 of SEQID NO:2). FIG. 3 demonstrates that difopein can bind 14-3-3 in vivo andcan disrupt the interaction of 14-3-3 with Raf-1. Specifically, HEK293cells were transfected with DNA coding for FLAG-tagged 14-3-3ζ andeither EYFP or EYFP-difopein as indicated. 14-3-3 was immunoprecipitatedusing anti-FLAG (M2; Sigma Chemical Co.). In the presence of EYFP,14-3-3ζ was able to co-precipitate endogenous Raf-1. However,EYFP-difopein blocked co-precipitation of Raf-1 and instead was itselfpulled down by 14-3-3. Thus, difopein can bind 14-3-3 in vivo and thisbinding can competitively disrupt the binding of 14-3-3 to ligands knownto interact in the amphipathic groove of 14-3-3. See also, Masters etal. (2001) Journal of Biological Chemistry 276: 45193-45200, hereinincorporated by reference.

EXAMPLE 2 Disruption of 14-3-3/Ligand Interactions Decreases CellViability

[0084]FIG. 4 shows the result from an attachment based viability assayin COS-7 cells. R18 is placed under the control of the CMV promoter in amammalian expression vector pCR3.1 (pSCM110). Cells expressing thecontrol pcDNA3 along with a lacZ marker gave high β-gal activity thatwas set as 100% survival. Expression of R18 drastically decreased thepopulation of viable cells, as shown by diminished β-gal activity. TheR18 effect was dose-dependent because increased R18 expression wascorrelated with decreased cell viability. Because of the high affinityof R18 for the 14-3-3 polypeptide and the interaction of R18 at theamphipathic binding groove, the decreased cell viability is likely dueto R18 induced disruption of 14-3-3 ligand interactions. This datasupports the notion that 14-3-3/ligand interactions are essential forcell survival. It appears that R18 induces cell death by an annexinV-positive phenotype, an early marker for apoptosis (data not shown).

[0085]FIG. 5 demonstrates cell death induced by the R18 antagonist ofSEQ ID NO:1 is dependant on acidic 14-3-3 interacting residues. The twoacidic residues of R18 (Asp and Glu) which are involved in coordinatingthe basic cluster of 14-3-3 were changed to lysine. This peptide is notexpected to bind 14-3-3, but it could retain interaction with othercellular partners if they do exist. Cos-7 cells were cotransfected witha farnesylated EGFP marker along with EYFP, EYFP-R18, or EYFP-R18(D12K,E14K). After 24 hours, cells were fixed in ethanol and DNA wasstained with propidium iodide. EGFP and PI signal intensities weremeasured on a FACScan flow cytometer (Becton Dickinson), and transfectedcells were placed in various phases of the cell cycle based on their DNAcontent. R18 caused an increase in the fraction of cells containing lessthan normal amounts of DNA (sub G₀), which is consistent with DNAfragments caused by apoptosis. R18 (D12K, E14K), which has chargereversals at two of the residues known to interact with 14-3-3, wasunable to induce cell death, which argues that the R18 induced death wascaused by its ability to bind and inhibit 14-3-3.

[0086] The second approach used a 14-3-3 binding molecule that isstructurally unrelated to difopein, which should therefore have adifferent spectrum of non-14-3-3 specific protein interactions. We havecharacterized the phosphorylation-independent interaction of 14-3-3 withPseudomonas aeruginosa exoenzyme S finding that it binds in theamphipathic groove of 14-3-3 with high affinity. See, for example,Masters et al. (1999) Biochemistry 38:5216-5221 and Zhang et al. (1997)J. Biol. Chem. 272:13717-13724. Others later determined that the 14-3-3binding epitope of exoenzyme S is localized to its C-terminal 54residues (Henriksson et al. (2000) Biochem 349:697-701). This domain,termed C54, was able to bind 14-3-3 and induce apoptosis in COS-7 cellsas determined by DNA content (data not shown). Thus, a strongcorrelation exists between the abilities of these molecules to bind14-3-3 and to cause cell death. This argues that the apoptosis caused bydifopein is due to inhibition of 14-3-3/ligand interactions and that14-3-3, by binding other cellular proteins, mediates a criticalprosurvival signal.

[0087] Studies further demonstrate that the difopein antagonist isdistributed through out the cell. EYFP or EYFP-difopein was transfectedinto Cos-7 cells. Approximately 24 hours after transfection cells werefixed with 4% paraformaldehyde and visualized by fluoresence microscopy.As shown in FIG. 6, EYFP-difopein was found throughout the cells and itsdistribution appeared identical to that of EYFP. Cos-7 cells wereassayed for cell death using EYFP-tagged R18 or a dimerized form of R18(difopein). Because 14-3-3 exists in vivo as a dimer, dimerization of14-3-3 ligands is expected to increase their affinity for 14-3-3. Asshown in FIG. 7, difopein dramatically enhanced the cell death ascompared to monomeric R18, and thus it was used for many of thesubsequent studies.

[0088] Further studies were preformed to demonstrate that difopeininduces cell death. In FIG. 8a, Cos-7 cells were co-transfected withmyc-tagged difopein or a control vector along with a lacZ reported gene.After approximately 48 hours floating cells were gently washed away andattached cells were lysed and assayed for β-galactosidase. Decreasedβ-gal activity indicated a loss of viability. As shown in FIG. 8a,expression of difopein decreased β-gal activity, an indication of a lossof cell viability.

[0089] In another assay, Cos-7 cells were cotransfected with a lacZmarker in addition to any EYFP-difopein fusion, EYFP alone, or emptyvector. Approximately 24 hours post transfection, cells were fixed andtransfected cells were identified by staining with X-gal. Viability ofthe transfected cells was determined by microscopic examination of cellmorphology: live cells were flat, while dead cells were rounded up. Asshown in FIG. 8b, EYFP expression had no effect on viability, but theEYFP-difopein fusion reduced the live cell population by about one-half

[0090] In yet another assay, (FIG. 8c) Cos-7 cells were transfected withEYFP or EYFP-difopein as indicated, and cell death was determined as inFIG. 5. Internucleosomal DNA cleavage, which is a commonly used markerof apoptosis, produces small fragments that can diffuse out of cellsduring ethanol fixation resulting in decreased signals being seen onstaining for DNA (Sub-G₀ DNA content). Therefore, the appearance of cellcontaining sub G₀ levels of DNA indicates apoptotic cell death. When subG₀ cells were excluded from analysis (not shown), no significantdifference in DNA content distribution was seen between EYFP andEYFP-difopein, suggesting that difopein does not disrupt the cell cycle.

[0091] It is possible that the effects of 14-3-3 inhibition on apoptosisare restricted to COS-7 cells. Three additional cell lines were used toexamine this issue: A549 lung cancer cells, DU145 prostate cancer cells,and HeLa cervical carcinoma cells. In the attachment-based viabilityassay, Myc-difopein was found to kill all three of these cell lines tovarying degrees (data not shown), supporting the generality of thedifopein effect. Because 14-3-3 proteins are ubiquitously expressed andbecause 14-3-3 can potentially target many different apoptosisregulating molecules, we anticipate that most cells will show somedegree of sensitivity to 14-3-3 inhibitors.

[0092]FIG. 9 demonstrates that difopein kills cells through an Aktindependent pathway. Cos-7 cells were assayed as in FIG. 5 except that avector expressing a constitutively active form of Akt was included.Difopein induced cell death is relatively insensitive to Akt expression.The Akt proto-oncogene acts on several targets to prevent cell death.This result supports the idea that difopein can be used to treat cancercells that have lost their sensitivity to apoptosis as a result ofincreased activity of prosurvival signaling networks.

[0093]FIG. 10 demonstrates difopein induced cell death is caspasedependent. Cos-7 cells were lysed 12 hours after transfection withvarious amounts of DNA coding for EYFP-difopein. Equal amounts oflysates were assayed for the ability to liberate p-nitroaniline (pNA)from the caspase 3 substrate Ac-DEVD-pNA. As shown in FIG. 10a,expression of EYFP-difopein led to a dose dependent increase in pNArelease that was completely blocked by the caspase 3 inhibitorAc-DEVD-CHO. In another study, Cos-7 cells were assayed as in FIG. 5,except that they were treated with 50 μM of the pan-caspase inhibitorzVAD-fink or vehicle immediately prior to transfection (FIG. 10b). Ascaspases are critical downstream effectors of numerous apoptoticstimuli, these results strongly support the hypothesis that difopeinkills cells through apoptosis.

EXAMPLE 3 14-3-3 Antagonists Sensitize Tumor Cells to Cisplatin-mediatedCell Death

[0094] Inhibition of 14-3-3/ligand interactions may facilitate apoptoticcell death induced by cytotoxic agents. To test this possibility, theeffects of expression of R18 and difopein on cisplatin-induced celldeath was examined (FIG. 11). In FIG. 11a, Cos-7 cells were transfectedwith a lacZ marker gene along with myc-tagged R18 DNA (amount used shownas the fraction of total DNA). 12 hours after transfection, theindicated concentrations of cisplatin were added. Approximately 36 hourslater the viability of the cells was determined as in FIG. 3 above. InFIG. 11b, HeLa cervical cancer cells were transfected with EYFP orEYFP-difopein as indicated. 24 hours later, 50 μM cisplatin or vehiclewas added. After 24 hours of cisplatin treatment, the cells were assayedas in 2 above. These data shown in FIGS. 11a and b demonstrate that theR18 and difopein 14-3-3 antagonist can enhance the ability of anantineoplastic drug to kill cells.

[0095] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0096] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 3 1 20 PRT Artificial Sequence R18 polypeptide sequence 1 Pro His CysVal Pro Arg Asp Leu Ser Trp Leu Asp Leu Glu Ala Asn 1 5 10 15 Met CysLeu Pro 20 2 62 PRT Artificial Sequence difopein polypeptide sequence 2Ser Ala Asp Gly Ala Pro His Cys Val Pro Arg Asp Leu Ser Trp Leu 1 5 1015 Asp Leu Glu Ala Asn Met Cys Leu Pro Gly Ala Ala Gly Leu Asp Ser 20 2530 Ala Asp Gly Ala Pro His Cys Val Pro Arg Asp Leu Ser Trp Leu Asp 35 4045 Leu Glu Ala Asn Met Cys Leu Pro Gly Ala Ala Gly Leu Glu 50 55 60 3 6PRT Artificial Sequence phosphoserine consensus binding sequence 3 ArgSer Xaa Ser Xaa Pro 1 5

That which is claimed:
 1. A method of enhancing the death of aneoplastic cell comprising providing to said neoplastic cell atherapeutically effective amount of a 14-3-3 antagonist and at least oneantineoplastic therapeutic agent.
 2. The method of claim 1, wherein said14-3-3 antagonist disrupts an interaction between a 14-3-3 polypeptideand a 14-3-3 ligand.
 3. The method of claim 2, wherein the 14-3-3antagonist directly interacts with a 14-3-3 polypeptide.
 4. The methodof claim 3, wherein the 14-3-3 antagonist is a 14-3-3 amphipathic groovebinding antagonist.
 5. The method of claim 1, wherein saidantineoplastic therapeutic agent is a chemotherapeutic agent.
 6. Themethod of claim 5, wherein said chemotherapeutic agent is selected fromthe group consisting of etoposide, cisplatin, doxorubicin, paclitaxel,methotrexate, and 5-fluorouracil.
 7. The method of claim 1, wherein saidantineoplastic therapeutic agent is a radiation.
 8. The method of claim1, wherein said 14-3-3 antagonist comprises an amino acid sequence setforth in SEQ ID NO:1, 2, or a biologically active variant thereof. 9.The method of claim 8, wherein said 14-3-3 antagonist comprises an aminoacid sequence selected from the group consisting of: a) an amino acidsequence having at least 80% sequence identity to the sequence of SEQ IDNO:1 or 2, wherein said sequence disrupts the interaction between a14-3-3 polypeptide and a 14-3-3 ligand; and, b) an amino acid sequencehaving at least 10 contiguous amino acids of SEQ ID NO:1 or 2, whereinsaid sequence disrupts the interaction between a 14-3-3 polypeptide anda 14-3-3 ligand.
 10. The method of claim 1, wherein the 14-3-3antagonist and the antineoplastic therapeutic agent are provided to thecell sequentially.
 11. The method of claim 1, wherein the 14-3-3antagonist and the antineoplastic therapeutic agent are provided to thecell simultaneously.
 12. The method of claim 1, wherein said neoplasticcell is in a mammal.
 13. The method of claim 13, wherein said mammal isa human.
 14. A method of treating a neoplastic disorder comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of a 14-3-3 antagonist and at least one antineoplastictherapeutic agent.
 15. A pharmaceutical composition comprising atherapeutically effective amount of a 14-3-3 antagonist and at least oneantineoplastic therapeutic agent.
 16. The pharmaceutical composition ofclaim 15, wherein said antineoplastic therapeutic agent is achemotherapeutic agent, a biological response modifier, or a radiation.17. The pharmaceutical composition of claim 16, wherein saidchemotherapeutic agent is selected from the group consisting ofetoposide, cisplatin, doxorubicin, paclitaxel, methotrexate, and5-fluorouracil.
 18. The pharmaceutical composition of claim 15, whereinsaid 14-3-3 antagonist disrupts an interaction between a 14-3-3polypeptide and a 14-3-3 ligand.
 19. The pharmaceutical composition ofclaim 18, wherein the 14-3-3 antagonist directly interacts with a 14-3-3polypeptide.
 20. The pharmaceutical composition of claim 19, wherein the14-3-3 antagonist is a 14-3-3 amphipathic groove binding antagonist. 21.The pharmaceutical composition of claim 20, wherein said 14-3-3antagonist comprises an isolated polypeptide comprising an amino acidsequence of SEQ ID NO:1, 2, or a biologically active variant thereof.22. The pharmaceutical composition of claim 21, wherein said polypeptidecomprises an amino acid sequence selected from the group consisting of:a) an amino acid sequence having at least 80% sequence identity to thesequence of SEQ ID NO:1 or 2, wherein said sequence disrupts aninteraction between a 14-3-3 polypeptide and a 14-3-3 ligand; and, b) anamino acid sequence having at least 10 contiguous amino acids of SEQ IDNO:1 or 2, wherein said sequence disrupts an interaction between a14-3-3 polypeptide and a 14-3-3 ligand.
 23. A method of identifying anagent that selectively inhibits an interaction between a 14-3-3polypeptide and a 14-3-3 ligand comprising: a) contacting a 14-3-3polypeptide with a 14-3-3 antagonist under conditions that permitformation of a 14-3-3/antagonist complex, said 14-3-3 antagonistcomprises a polypeptide having an amino acid sequence selected from thegroup consisting of: i) an amino acid sequence set forth in SEQ ID NO:2or a biologically active variant thereof; ii) an amino acid sequencehaving at least 80% sequence identity to the sequence of SEQ ID NO:2,wherein said sequence disrupts an interaction between a 14-3-3polypeptide and a 14-3-3 ligand; and, iii) an amino acid sequence havingat least 10 contiguous amino acids of SEQ ID NO:2, wherein said sequencedisrupts an interaction between a 14-3-3 polypeptide and a 14-3-3ligand; b) contacting said 14-3-3/antagonist complex with a candidateagent; and, c) determining if the candidate agent disrupts the14-3-3/antagonist complex.
 24. A method of identifying an agent thatselectively inhibits an interaction between a 14-3-3 polypeptide and a14-3-3 ligand comprising: a) contacting a 14-3-3 polypeptide with acandidate agent under conditions that allow for a 14-3-3/candidate agentcomplex to form; b) contacting said 14-3-3/candidate agent complex witha 14-3-3 antagonist, said 14-3-3 antagonist comprises a polypeptidehaving an amino acid sequence selected from the group consisting of: i)an amino acid sequence set forth in SEQ ID NO:1, 2, or a biologicallyactive variant thereof; ii) an amino acid sequence having at least 80%sequence identity to the sequence of SEQ ID NO:1 or 2, wherein saidsequence disrupts an interaction between a 14-3-3 polypeptide and a14-3-3 ligand; and, iii) an amino acid sequence having at least 10contiguous amino acids of SEQ ID NO:1 or 2, wherein said sequencedisrupts an interaction between a 14-3-3 polypeptide and a 14-3-3ligand; and, c) determining if the 14-3-3 antagonist disrupts the14-3-3/candidate agent complex.
 25. The method of claim 23 and 24,wherein said method occurs in vivo or in vitro.
 26. An isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of: a) an amino acid sequence set forth in SEQ ID NO:2; b) anamino acid sequence having at least 80% sequence identity to thesequence of SEQ ID NO:2, wherein the sequence disrupts the interactionof a 14-3-3 ligand with a 14-3-3 polypeptide; and, c) an amino acidsequence having at least 19 contiguous amino acids of SEQ ID NO:2,wherein the sequence disrupts the interaction of a 14-3-3 ligand with a14-3-3 polypeptide.